The research interests of this laboratory are focused on understanding the structure and functions of proteins encoded by gene 1 of the coronavirus mouse hepatitis virus (MHV-A59). The mouse hepatitis viruses are important models for the study of human demyelinating neurological diseases such as multiple sclerosis, as well as for investigation of the molecular biology of coronavirus replication. Although transcription and replication of the coronavirus genome has been investigated in depth, much less is known about the gene 1-encoded nonstructural proteins predicted to account for these activities. Gene 1 comprises the 5' two- thirds of the 32kb MHV genome RNA and potentially encodes 800 kDa of polypeptide in its two overlapping reading frames. Gene 1 contains conserved RNA sequences, predicting proteins with strong similarity to proteinase, replicase, and helicase proteins of other RNA viruses. Gene 1 also contains large regions of sequence(10kb) encoding polypeptides for which there are no known viral or cellular homologs. We have thus far characterized in MHV infected cells the translation and proteolytic processing of the N-terminal 380 kDa of gene 1-encoded products. The principal focus of this project will be to complete the description of the all of the distinct precursors and products encoded in gene 1. Antibodies directed against polypeptides encoded in gene 1 will be used to characterize the intracellular translation, processing, alignment, and N-terminal amino acid sequence of gene 1 proteins in MHV-A59 infected murine cell monolayers. The data on intracellular expression and processing will be utilized to investigate translation of gene 1 in cell- free lysates, in order to more specifically define the effects of proteinase inhibitors, cellular proteins, and membranes on the expression and processing of gene 1 proteins. Since reticulocyte lysates supplemented with cell extracts. The data obtained from these experiments will be used in the initial functional studies of gene 1 products, which will focus on the predict protease activities precursor proteins, both in cells and in cell-free lysates, with concomitant inhibition of virus RNA synthesis and inhibitors leupeptin and E64d. Gene 1 polyprotein processing of proteinase inhibitor-resistant various will compared with that observed in nonresistant parental MHV-A59 strains, and the sequences of predicted proteinase domains from resistant variants will be determined. These variants will allow us to define the regions of gene 1 important for proteolytic processing. This systematic investigation of gene 1 translation and processing will provide important insights into mechanisms of coronavirus replication, as well as identifying potential targets for antiviral intervention. The data will also further our long-term goal of expressing and purifying individual proteins of MHV gene 1, in order to define their unique functions and interactions during coronavirus replication.